Studies on the in vitro metabolism of 25-hydroxyvitamin D3 (25(OH)D3) by both renal and extrarenal cells has extended our knowledge of the vitamin D3 endocrine system. For the most part these types of experiments have utilized isolated cells in either monolayer culture or in suspension. At present, a number of cell types have been assayed for 25-hydroxyvitamin-D3-1- and 24-hydroxylase (1-hydroxylase and 24-hydroxylase) activity. Cells isolated from kidney, bone, intestine, placenta, decidua and chorioallantoic membrane appear to be able to produce both 1,25(OH)2D3 and 24,25-dihydroxyvitamin-D3 (24,25(OH)2D3) from 25-hydroxyvitamin D3 (25(OH)D3) and cells isolated from liver, skin, skeletal muscle, heart muscle, cartilage, etc. have no such activity (except for the possible synthesis of 24,25(OH)2D3 by cartilage). These observations have led to the proposition of a new hypothesis which states that all tissues involved in homeostasis of calcium and phosphorous can synthesize at least small amounts of 1,25(OH)2D3, probably for local use. In general it is the aim of this proposal to further test this hypotesis by characterizing the ability of extrarenal cells, especially bone cells, to synthesize 1,25(OH)2D3 and 24,25(OH)2D3. Specifically, experiments will be performed to determine 1) the effect of agents known to mediate mineral homeostasis on bone cell 1- and 24- hydroxylase activities, 2) the effect of age and state of differentiation of bone cells on 1- and 24-hydroxylase activities, 3) the biological effectiveness of the 1,25(OH)2D3 synthesized b bone cells, and 4) other cell types which can synthesize 1,25(OH)2D3 and 24,25(OH)2D3 and may conform to the above stated hypothesis. The methodology will include all of the techniques for the isolation and culture of bone and other cell types as well as those for the separation, identification and metabolism of vitamin D3 metabolites. If these goals can be achieved the ensuing data should support the physiological importance of extrarenal 1,25(OH)2D3 synthesis and may change the way we view the integrated control of mineral metabolism.